Reagent material and method for creative kinase assay



" f nited States Int. Cl. G01n 31/14 US. Cl. 195-1035 14 Claims ABSTRACT OF THE DISCLOSURE Substantially anhydrous, solid assay materials for the determination, inter alia, of Regent for Assaying Creatine Phosphokinase are rendered storage stable by the presence of certain polyhydric compounds preferably mannitol, sorbitol, lactose or polyvinyl alcohol.

This application is a divisional of my copending application Ser. No. 561,757, filed June 30, 1966, now US. Pat. 3,413,198, which in turn is a continuation-in-part of my copending application Ser. No. 320,004, filed Oct. 30, 1963, and now abandoned.

The present invention relates to processes and compositions for preparaing reagent mixtures for detecting and measuring the presence of certain components in a biological sample. It also relates to the novel reagent mixtures.

In the clinical diagnosis of certain pathological conditions, it is frequently valuable to know the amount of activity or the quantity of certain substances present in a specimen of a biological or other fluid or tissue. One of the more effective means that has been proposed for making assays of such specimens is to provide a liquid reagent which contains one or more biological components. When a given reagent is mixed with the specimen, the components are effective to cause an enzymatic reaction that invOlVes the unknown substance. By observing this reaction, it is possible to determine the quantity or amount of activity of the unknown originally present.

Since such reagents contain one or more biological components such as enzymes, coenzymes and/or substrates, etc., the reagent has inherently been of a very unstable nature and has very little if any shelf life. To insure the reagent being at optimum strength it must be preparaed at or immediately prior to the time the assay is made. In addition, heretofore the various components such as the enzymes, coenzymes, substrates, etc., included in the reagent have been very unstable. To insure these components being at their optimum it has been necessary for the components to be stablized in a concentrated form.

When it has been desired to make a biological assay or the present type, a kit containing the several different components which may be dry, or in solutions, has been obtained. If the components are in a dry form, aqueous solutions are formed, and maintained separately until just prior to use.

' The various components for the reagent are present in separate containers and maintained separated from each other. Some of these solutions and particularly those containing the enzymes are necessarily in a concentrated form in order to preserve their activity.

When employing a kit of this type, to assay a specimen, it is necessary to first reconstitute the components to the required strength by adding a specified amount of icev another liquid such as water to various solutions. After all of the various components have been reconstituted, the appropriate quantities of each are combined to form the reagent. A predetermined quantity of the reagent is then mixed with the specimen to produce the desired assay reactions. The accuracy of the final assay is also dependent upon the accuracy with which components are reconstituted, the accuracy with which the reconstituted components are combined to form the resultant reagent and the accuracy with which the reagent is measured when it is mixed with the specimen. It may thus be seen that the accuracy of the assay is dependent upon the skill of the operator and the accuracy with which he prepares and uses the reagent.

It can be readily appreciated that the foregoing porcess is very time-consuming particularly when considering the time for using and cleaning the substantial amounts of equipment such as various pieces of glassware, measuring instruments, etc. If any of the equipment has any foreign matter thereon, the reagent may easily be contaminated whereby the results of the assay will be misleading.

It should also be noted that after the reagent is fully prepared, at least one of the components therein and particularly the enzymes are quite unstable and rapidly lose their activity. As a consequence, if the reagent is not used within a matter of a few hours following its preparation, it must be discarded and, therefore, wasted. The percentage of the reagent wasted in this manner hecomes very large where only a few assays are made at infrequent intervals.

It may thus be seen that although the foregoing kits have been capable of producing the desired reactions and permitting the desired assays to be made they have not been entirely satisfactory for numerous reasons. For example, they have not only been very time-consuming and wasteful, but have also required a person of sufficient skill to insure the accurate preparation of the reagents and their being used in the proper manner. Also, because of the possibility of substantial human errors such reagents have induced a certain degree of unpredictable error in the results of the assay.

It is an object of the present invention to provide means which will be effective to overcome the foregoing difliculties. More particularly, it is proposed to provide new and novel assay materials useful in making biological assays and the method for preparing the materials. All of the assay materials are in a dry, solid state that may be easily handled and used. The assay materials include components such as enzymes, coenzymes and/or substrates which have heretofore been very unstable. Moreover, the combining of such components tends to reduce their stability. However, stabilizers are included that are effective to maintain or preserve the activity of each of the components and of the entire assay material. Each of the components including those containing the enzymes may be stabilized individually and used as such for any desired purpose. Also, the compounds may be combined together to form a new and novel assay material. The resultant assay material contains all of the com ponents except water, for making a liquid reagent that can be used to make a biological assay of the above described type.

Although the assay material contains various components such as enzymes, coenzymes and/ or substrates, etc., the material is in a dry, solid form and will be very stable and have a long shelf life. This will permit the assay material to be packaged into containers which are easy to handle and use. Each of the containers may include a quantity of the assay material that is just the right amount for making a particular number of assays, for example a single assay. The assay material may desirably include an agent which, among other things, is effective to increase the volume of the assay material to a standard size whereby the quantity of the assay material required to produce a single assay will always be a standard amount. In order to make an assay, the contents of one of a standard size container may be mixed with a predetermined quantity of water to produce a liquid reagent that is suitable for making a single assay. In addition this agent can facilitate handling during manufacture and increase shelf life. A preferred agent is mannitol. It will thus be seen that the possibility of human errors is eliminated and a relatively inexperienced person may prepare the liquid reagent and make the desired assay without any time-consuming measurements, using any large quantities of glassware, etc.

The term enzymatic assay is defined as the use of an enzyme as a component of a reagent for the determination of a substance or another enzyme, or for the determination of an enzyme in a biological sample.

These and other objectives and advantages of the present invention will become readily apparent from the following detailed description of a limited number of embodiments of the persent invention.

The present invention is particularly adapted to be embodied in a dry assay material for determining in a specimen of fluid, particularly of biological origin, the quantity or the amount of activity of a certain unknown.

In the present instance, the unknowns are in a class which for convenience may be divided into four separate groups. The groups include enzymes such as carboxylases, dehydrogenases, hydrolases, isomerases, oxidases, phosphorylases and transferases. By way of example, this group includes: lactate dehydrogenase, alkaline phosphatase, glu cose oxidase, muscle phosphorylase, glutamate-oxaloacetate transaminase, phosphoenol-pyruvate carboxylase, cholinesterase, glutamate-pyruvate transaminase, malate dehydrogenase, acid phosphatase, prostatic acid phosphatase, esterase, diesterase, lipase, amylase, sorbitol dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, aldolase, glutamate decarboxylase, uricase, galactowaldenase, triose phosphate isomerase, carbonic anhydrase, leucine aminopeptidase, 3-phosphoglyceraldehyde dehydrogenase, trypsin and chymotypsin. Also included are kinases, like creatine kinase.

The second group includes biochemical intermediates or metabolites. By way of example, the second group includes: glucose, lactic acid, pyruvic acid, adenosine triphosphate, phenylpyruvic acid, 3 methoxy-4-hydroxymandelic acid, cholesterol, creatinine, creatine, urea, uric acid, aspartic acid and glycine.

The third group includes chemical constituents of cells or biological fluids which, by way of example, may include dissolved carbon dioxide, triglycerides, protein, starch, glycogen, hemoglobin and insulin.

The fourth group includes drugs and toxins such as antimycin A, diisopropylfluorophosphate, sul-fathiazole, ethanol, acetaldehyde and barbiturates.

To assay a specimen for one of the unknowns within a class, a liquid reagent may be mixed with the specimen to produce an enzymatic reaction. The particular reaction that occurs should produce an efiect which can be easily measured. By way of example, the optical density of the assay mixture at some predetermined wavelength may change in proportion to the extent of the reaction.

In the present instance, the liquid reagent is prepared by dissolving a dry, solid assay material in water. When this solution is mixed with the specimen an assay mixture will be formed that includes a substrate that will enter into the reaction, an enzyme that will catalyze the reaction, and a coenzyme that will be oxidized or reduced in the course of the reaction so as to produce a desired change in the assay mixture, for example, its optical density. All of the components that are not present in the specimen are contained in the assay material. In addition, one or more substances are included in the assay material to stabilize the assay material and preserve the activity of the various components. In addition, one or more buffer substances may also be provided that will be effective to maintain the conditions in the assay mixture suitable for the reaction to occur at an optimum rate.

The assay material is in a dry, powdered form and contains all of the components except water, for producing an enzymatic assay when combined with the specimen.

Since the assay material is very stable, the powder may be pre-measured into portions which are of just the right amounts for producing an assay reaction in a single specimen or an integral number of specimens. The pre-measured quantity of the assay material may be dissolved directly into a suitable quantity of water to form a liquid reagent. The liquid reagent may then be mixed with the specimen to induce the assay reaction in the assay mixture.

If the unknown being assayed is an enzyme, the reagent will not necessarily include an enzyme. Accordingly in one embodiment, the assay material is free of any enzymes but includes one or more components such as a substrate that will react at a rate or to the extent that is determined by the amount of activity of the unknown or enzyme originally present in the specimen.

In a second embodiment of the assay material, a substrate is included which will react with the unknown and an enzyme included in the reagent, that will catalyze the reaction. In order to prepare an assay material of this embodiment, the first step is to select one or more substrates and one or more enzymes that will be efiective to produce an enzymatic assay reaction and insure its occurring in the desired manner. The particular enzymes that are chosen will, of course, depend upon the particular unknown to be assayed and the particular reaction which it is desired to create. However, the enzymes will normally be chosen from a class that includes carboxylases, dehydrogenases, hydrolases, isomerases, oxidases, phosphorylases, and transferases. By way of example, this class includes: lactate dehydrogenase, alkaline phosphatase, glucose oxi dase, muscle phosphorylase, glutamate oxaloacetate trans aminase, phosphoenolpyruvate carboxylase, chlorinesterase, glutamatepyruvate transaminase, malate dehydrogenase, acid phosphatase, prostatic acid phosphatase, esterase, diesterase, lipase, amylase, sorbitol dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, aldolase, glutamate, decarboxylase, uricase, gelactowaldenase, triose phosphate isomerase, carbonic anhydrase, leucine aminopeptidase 3-phosphoglyceraldehyde dehydrogenase, trypsin, chymotrypsin, alpha-hydroxybutyrate dehydrogenase and beta-hydroxybutyrate dehydrogenase.

As is well known, enzymes of this type are generally very unstable. In fact, heretofore, in order to maintain enzymes of this type in a stable condition, it has been necessary to keep them in a concentrated form and at low temperatures. In addition to being concentrated, it has also been usually necessary for the enzyme solution or suspension to include a substantial amount of salt such as ammonium sulfate to maintain optimal activity.

In the present invention, to prepare the assay material in a dry form, one of the steps in the process is to convert the enzyme from a solution to a dry, solid form such as a powder in which the enzyme is very stable. To accomplish this, one or more stabilizing compounds may be added to the solution containing the enzymes. The particular stabilizers added to the enzyme solution will, in part, vary with the particular enzyme that is to be stabilized. However, for enzymes of this type, at least one stabilizer is chosen from one or more of the following groups. Under some circumstances it has been found to be advantageous to employ a combination of stabilizers which may include a stabilizer from several of the following groups or even a stabilizer from each group.

Group I.Mucilagenous gums or polysaccharides such gum acacia, gum caarageenan, tragacanthin, alginic acid and pectin substances. Gum acacia has been found to be particularly well suited for this purpose. In addition to or as an alternative to the gums, the stabilizer may include other polymers containing hydroxy groups or other hydrophilic substitute groups which render the resultant polymer essentially soluble in water such as, polyvinylpyrrolidine, Carbowax and polyvinyl alcohol. This will also insure all of the assay material dissolving rapidly in the water when the reagent is prepared. However, it is also possible to use any other polymers which because of large chains or hydrophlic substituent is only partially soluble in water but which equilibriate with the aqueous phase such as ion exchange resins, ion exchange cellulose, carboxymethyl cellulose.

Group II.-A buffer consisting of a hydroxylalkylamine including but not limited to primary amines such as tris (hydroxymethyl) aminomethane or a tertiary amine such as triethanolamine.

Group III.-A sequestering or complexing agent such as ethylene diamine tetracetic acid or one of its salts which has been found to be particularly well suited.

Group IV.An inert soluble protein such as bovine serum albumin.

Group V.,Salts of a polyvalent anion such as ammonium sulfate, or sodium potassium tartrate, which have been found particularly suitable.

Group VI.Sulfhydryl compound such as dithioerythritol, cysteine, or reduced glutathione, which have been found particularly suitable.

After one or more of the stabilizers of the class described above has been completely dissolved or uniformly dispersed throughout the entire mixture. The enzyme or enzymes in the solution are very stable. It has been found that by adding these stabilizers to the solution, the activity of the enzymes is often increased. This is believed to result from the elimination of the effects of certain inhibitors which are usually present with the enzymes. In the event it is desired to decrease the activity of the solution, the solution may be diluted by adding water. Conversely, if it is desired to concentrate or increase the amount of activity of this enzyme solution, a portion of the liquid may be removed. Preferably, the liquid is removed by evaporation while the solution is maintained at a relatively low temperature. It may thus be seen that a very stable enzyme solution is provided at this point and that the stability of the solution is independent of the concentration of the enzyme or the salts therein and exhibits stability over a wider range of temperature.

It is an overall objective to provide an integrated assay material, which is dry, stable, enzymatic, pyridine nucleotide, linked, and uniform in results under varying climatic and storage conditions.

The above described stabilized enzyme solution may be used for numerous purposes as a solution. However, under some circumstances such as the preparation of the present assay material, it may be desirable to convert the solution into a dry mixture or powder containing the enzyme. This may be accomplished by lyophilizing or freeze drying the solution. More particularly, the entire solution is frozen to provide a solid mass and placed under a vacuum. The vacuum is of suflicient magnitude to cause the frozen liquid to sublimate. The frozen mass is kept under this vacuum for a period sufficient to insure all of the water, etc., being removed. This will leave a solid residue that contains the enzyme or enzymes in intimate relation with the stabilizers, such as acacia, etc. These stabilizers are effective in preserving the activity of the enzyme for an extended period of time even though the enzyme is in the form of a solid.

The term stabilizer, as used herein, broadly relates to a substance which prevents the change or destruction of a reagent component. It has three major aspects in the present invention, namely: (1) to allow for convenient handling of the components during manufacture; (2) to permit the preparation and storage of a component in dry form; and (3) to provide longterm shelf-life of the finished product.

The residue will normally be in a fluffy or flaky condition. However, if it is desired, the residue may be ground until it is reduced to a finely powdered mixture. The grinding may be accomplished by any suitable means such as a ball mill. The fact that the powder mixture is substantially dry contributes to the chemical stability of the enzymes. It also contributes to the physical stability of the mixture and substantially eliminates the tendency to compact or become lumpy, etc. Since the mixture can be maintained as a loose powder it will be easy to handle and process. Also, it can be easily measured either volumetrically or gravimetrically. At this point a powder is provided that includes one or more enzymes in a very stable form. Since the powder does not include any form of substrate, it can be used as a enzyme for any desired purpose. For example, among other things, the enzyme powder may be employed to complete the preparation of the present assay material.

In order to complete the preparation of the assay material the other components such as the buffers, substrates, coenzymes, and bulking-stabilizing agents, may be prepared for mixing with the stabilized enzymes. It is one of the primary purposes of the buffer materials to maintain the conditions suitable for the assay reaction to occur at an optimum rate. When the assay material is dissolved in water, the buffers will, among other things, be effective to maintain the pH of the liquid reagent. In addition, when the liquid reagent is, in turn, mixed with a specimen containing the unknown, the pH of the resultant specimen mixture will still be suitable for the assay reaction to occur.

The particular buffer material that is employed in any particular assay material will be dependent upon the particular assay reaction to be conducted and the other components in the assay material. However, normally, they will be in a class that includes the salts of polyvalent inorganic anions and organic amines together with the acids and salts thereof. By way of example, the salts of polyvalent inorganic anions may include at least sodium and potassium phosphates and sodium and potassium pyrophosphates. By way of example, organic amines and acids, and their salts may include at least tris (hydroxymethyl) amino-methane and imidazole (and their salts, such as the hydrochloride, succinate, sulfate), succinic, aspartic, and glutamic acids (and their salts such as the sodium, potassium, and lithium), glycylglycine, and glycine.

The buffer materials may be prepared in the form of a dry powder that is mixed directly with the lyophilized powder containing the enzyme and the stabilizer. The resultant powder will contain the enzymes required for the assay reaction. Because of the stabilizers and buffers present in the powder, the enzymes will be very stable. This resultant powder like the enzyme powder first described, will not be hygroscopic, in contrast to corresponding mixtures prepared by lyophilization of the combined buffer and enzyme solutions.

The substrate is effective for reaction with the unknown in the specimen. Accordingly, the particular substrate that is employed in any particular assay material will be dependent upon the nature of the unknown and the particular assay reaction that it is desired to produce. Normally, the substrate will be in a class of biochemicals whose chemical reactions will be specifically catalyzed by the classes of enzymes previously described.

The following are specific examples of some of the substrates that fall within this class: Alanine, alphaor .7 beta-ketoglutaric acid, aspartic acid, fructose-1,6-diphosphate and glucose.

When the assaymaterial is dissolved to form a liquid reagent and the reagent is mixed with the specimen, the substrate will react with the unknown. However, in order for the reaction to occur successfully, it is necessary for the enzyme to catalyze the reaction. The quantity of the substrate and the amount of activity .of the enzyme contained in the reagent are in excess of that required to cause all of the unknown to completely react or to react at a desired rate. As a result the only factor that limits the assay reaction will be the quantity or amount of activity of the unknown.

When the substrates are in a pure solid dry form, they may be ground into a dry powder suitable for mixing with the lyophilized powder.

The coenzyme enters into the reaction and is converted from one form to another form. The extent to which the coenzyme is converted is determined by the extent to which the assay reaction progresses. The coenzyme may be readily converted from one form (such as oxidized) to another form (such as reduced). In addition the coenzyme has a light absorption at some particular wavelength only when it is in one of these forms. When it is in the other form, it is transparent at the designated wavelength, although the absorption band may be any desired wavelength that is convenient to use. However, it is desirable that it be distinct from the intense absorption bands of the rest of the components in the assay material and the substances in the specimen. This will insure all of the substances in the reagent and the specimen, except the coenzyme, being transparent or substantially transparent although some of the various components may absorb limited quantities of light in the region of the selected wavelength and they will not vary during the period of assay whereby the only variable will be the coenzyme in the absorbing form. Thus by measuring the optical density at the designated wavelength, the amout of the coenzyme converted may be determined. More specifically, by measuring the amount of change or rate of change of the optical density at the designated wavelength, the amount or rate of the assay reaction may be measured. It has been found that the pyridine nucleotides are particularly well suited for this purpose. When they are in their reduced form, they show absorption of ultraviolet light with a maximum value at a wavelength of about 340 millimicrons. By employing a coenzyme of this class in all forms of the assay material, the assay reactions may be observed by always measuring the optical density at this wavelength.

Coenzymes of this class have a limited amount of stability in a solid form. They can be stored ina solid form for only short periods of time. The stability of the coenzyme may be increased by preparing a lyophilized powder of the coenzyme and acacia. Further increases in the stability of the coenzyme may be obtained by mixing with mannitol. Accordingly, the coenzyme may be ground into a powder and mixed directly with the lyophilized powder containing the enzyme, stabilizer, and buffer.

Heretofore, substrates, enzymes and coenzymes, and buffers have been dissolved in the solution containing the enzyme before lyophilization. The complete solution may then be lyophilized to provide a dry residue containing all of the components of the assay. However, it has been found, as a practical matter, the resultant assay material is often hygroscopic. As a consequence, the residue sometimes tends to absorb varying or unpredictable quantities of moisture. As a result when prepared in this manner it is desirable for the resultant mixture to be hermetically sealed within a container. However, even when hermetically sealed, the mixture may still be unstable and tend to develop colored specks due to local decomposition and in a fairly short time completely decompose or lose its activity. It has also been found that the mixture tends to form into lumps which makes it difficult to handle and 8 measure into small units of identical amounts on a volumetric or Weight basis.

This invention teaches that by preparing the various components of the assay material such as the substrates and coenzymes in a dry or solid state, and in a stabilized form before the mixing thereof With the lyophilized stabilized enzyme, a much more stable and easily handled assay material is now provided.

In addition to the foregoing components, it has also been found desirable to add a bulking-stabilizing agent to the mixture. This agent may be a polyhydric substance such as mannitol, sorbitol, lactose, polyvinyl alcohol or polymers having from 1 to 5 hydroxyl groups per monomeric unit. The bulking agent is not active in the assay reaction. Accordingly, the quantity of the bulking agent added to the assay material is not critical and may be varied throughout a Wide range. However, the bulking agent performs several unexpected and useful functions. First of all, the bulking agent tends to further increase the stability of the assay material for several reasons. Such agents have the ability to absorb and retain limited quantities of moisture whereby the assay material is not materially affected when exposed to reasonable amounts of moisture. This increases the stability of the assay material and prevents its losing its activity. It has also been found that the bulking agent will also be effective in preserving the assay material by increasing the compatibility of its components. It has also been found that bulking agents of this category are also effective in increasing the ability of the assay material to withstand relatively high temperatures, such as 50 C., for longer periods of time. Heretofore, temperatures in this range have caused rapid deterioration of the enzymes, coenzymes, and other components.

Secondly, it has been found that the use of the bulking agent in the assay material results in the assay material dissolving more rapidly into water. This not only reduces the time required for preparing the liquid reagent but also increases the convenience of preparation by reducing the amount of stirring or shaking.

Thirdly, since the bulking agent does not enter into the reaction or affect the components in the assay material, the quantity of the bulking agent added to the assay material may vary over a wide range. Once a batch of the assay material has been prepared, its strength or amount of activity may be determined. The bulking agent may then be added to standardize the assay material to a predetermined level. This will result in the assay material always having a predetermined amount of activity per unit irrespective of the batch in which it is prepared. Of the agents listed above, mannitol is preferred.

After the assay material has had the bulking agent added, it may be divided into units of a standard predeter mined size. The size normally will be just large enough to make a single assay or an integral number of assays. Each of these units may then be packaged into a container such as a capsule, glass vial, etc.

It will thus be seen that a plurality of substantially identical packages such as foil containers or capsules may be provided. Each of these capsules will contain just a sufficient quantity of the assay material for making a single assay of a specimen. In order to make an assay, a package containing the assay material for making the particular assay is selected. The assay material contained in the package is all pre-measured and of a predetermined activity. Accordingly, it may be dissolved directly in a standard amount of water so as to form a liquid reagent. This liquid reagent is then mixed with the specimen to produce an enzymatic reaction. The extent of or the rate at which the reaction occurs will be a function of the quality or amount of activity of the original unknown. Every test, the irrespective of the particular type of assay, will involve the conversion of a coenzyme from one form to another form wherein one form has an optical absorption at a predetermined wavelength. Accordingly, the optical density of the specimen at that wavelength will vary as a function of the unknown. Thus, 'by measuring the optical density of the medium at different times, it will be possible to compute the quantity or amount of activity of the unknown in the original specimen.

The invention sought to be patented in a principal process of making aspect, is described as residing in the concept of preparing an assay reagent, useful, inter alia, as an aid in the clinical diagnosis of pathological conditions to determine the presence of, quantity of, or amount of activity of an enzyme in a biological specimen by mixing therewith a lyophilized, stabilized, catalytic reagent while in substantially anhydrous particulate form, comprising a dry coenzyme, a dry buffer, a dry second enzyme capable of catalyzing the conversion of the coenzyme to the other form of the coenzyme, dry substrates, effective to react with the enzyme of which the present quantity or activity is to be determined.

The invention sought to be patented in a second embodiment of a process of making aspect is described as residing in the concept of preparing an assay reagent, useful, inter alia, as an aid in the clinical diagnosis of pathological conditions to determine the presence of, quantity of, or amount of activity of creatine phosphokinase in a biological specimen by mixing therewith a lyophilized, stabilized, catalytic reagent while in substantially anhydrous particulate form including the combination of: a dry substrate comprising creatine phosphate; the dry enzymes hexokinase and glucose 6 phosphate dehydrogenase; a dry coenzyme comprising adenosine triphosphate and triphosphopyridine nucleotide a dry buffer capable of maintaining the pH between 6.7 and 6.9; at least one dry stabilizer selected from mucilagenous gums, hydroxyalkylamines, ethylenediamine tetraacetic acid and its salts, an inert soluble protein, a source of sulfate anion; a thiol compound and preferably dry activator; which involves determining the optical density of the solid reagent after forming a liquid reagent therefrom, and again following a predetermined incubation period after admixing the specimen.

The invention sought to be patented in a tenth composition of matter aspect is described as residing in the concept of a substantially anhydrous solid reagent useful, inter alia, as an aid in clinical diagnosis of pathological conditions to determine the presence of, quantity of, or amount of activity of creatine phosphokinase in a biological specimen by mixing therewith a lyophilized stabilized, catalytic reagent while in substantially anhydrous particulate form including the combination of: a dry substrate creatine phosphate; the dry enzymes hexokinase and glucose 6 phosphate dehydrogenase; the dry coenzymes adenosine triphosphate and triphosphopyridinenucleotide; a dry buffer capable of maintaining the pH between 6.5 and 7.5; at least one dry stalibilizer selected from mucilagenous gums, hydroxyalkylamines, EDTA and its salts, an inert soluble protein, a source of sulfate anion; a thiol compound; and preferably a dry activator.

EXAMPLE A An example of the first embodiment of this invention is a solid reagent that is particularly adapted to be employed for measuring the amount of activity of creatine kinase, creatine phosphokinase or CPK present in a biological sample. This reagent fully prepared will consist of a dry mixture of the following solid substance.

enzyme: hexokinase and glucose 6 phosphate dehydrogenase buffer: tris-(hydroxymethyl)aminomethane-succinate stabilizer: acacia tris (hydroxymethyl)aminomethanesulfate, ammonium sulfate and a thiol compound like cysteine substrates: creatine phosphate and glucose activator: magnesium sulfate accelerator: insulin coenzymes: adenosine diphosphate and TPN inhibitor: adenosine monophosphate.

In order to prepare a large number of capsules of this reagent the following procedure may be employed to produce a batch of dry reagent that may then be divided into small quantities and packaged into suitable capsules. Wherever quantities are specified in this example they are suitable for preparing a batch that will yield on the order of 10,000 capsules. It should be understood that whenever desired these quantities may be varied to satisfy other requirements.

The first step in this procedure is to prepare a suitable dry lyophilized powder containing the enzymes hexokinase and G-6-PDH, which may then be prepared by combining the following chemicals in the indicated ranges:

Gum acacia gm 50-100 Tris-sulfate, 0.2 M pH 7.4 to 7.6 ml -100 Ammonium sulfate, 1 M pH 7.5 (adjusted with ammonium) ml 75100 Insulin mg 50-750 Hexokinase mg Glucose 6 phosphate dehydrogenase mg 100 The tris-sulfate solution is prepared first by dissolving tris (hydroxymethyl) aminomethane and adding a suf ficient quantity of sulfuric acid to make a 0.2 molar solution with a pH 7.4 to 7.6.

The acacia, ammonium sulfate and insulin are completely mixed with the tris-sulfate to form a homogenous solution. After the solution is completely mixed any air entrapped therein may be removed by placing the solution under a vacuum. The enzymes hexokinase and glucose-fi-phosphate dehydrogenase are mixed into this solution. This solution should be thoroughly mixed to insure the enzymes being completely dispersed therein. When uniformly mixed, the solution is completely frozen. While in the frozen state it is placed under a vacuum until all of the moisture is removed. This will leave a homogenous dry powder containing the enzymes. This lyophilized powder may then be assayed to determine the amount of activity of the enzyme required by employing the following chemicals in the indicated amounts to form a reagent:

Glucose solution, 60 ,umoles/ml. (1.08 g. in 100 ml.) ml 0.5 Creatine phosphate, 100 mg./ml ml 0.2 Tris-succinate buffer solution ml 1.0 Adenosine diphosphate, l5 mg./ml. ml 0.2 Magnesium sulfate hydrated, 1 g./ 10 ml ml 0.4 Adenosine monophosphate, 60 mg./l ml. ml 0.2 TPN, 10 mg./1 ml. water ml 0.2 Water ml 0.4 Lyophilized enzyme mixture mg 10.0 Cysteine hydrochloride, 12.5 mg./ml. ml 0.2

The buffer employed is substantially identical to the tris buffer used in Example D of the parent case and consists of a mixture of tris (hydroxymethyl) aminomethane and succinic acid. These chemicals may be mixed in substantially the same manner as described in Example D to produce a dried buffer powder that when dissolved in water will produce a solution having a pH between 6.7 and 7.2.

Tris-succinate has been found to be a preferred buffer; however, any compound which can maintain the pH between 6.7 and 7.2, may be used, such as an alkali metal phosphate, or triethanol amine.

The tris succinate buffer solution is prepared by dissolving 3.4 grams of the buffer prepared as above described in 100 milliliters of water. Following this the in dicated quantities of the rest of the solutions are combined and mixed with the indicated quantity of Water. After the reagent has been completed its optical density at 340 millimicrons is measured. Following this a standardized creatine phosphokinase solution containing 0.02

1 1 International Enzyme Units 1 may be added to the mixture. When this occurs the reaction subsequently described will occur. The change with time of the optical density at 340 millimicrons is measured. By comparing the change in optical density the activity of CPK can be as result of the reaction be determined.

Under the foregoing test conditions one International Enzyme Unit of the CPK should produce an optical density change at 340 millimicrons of 2.07 per minute at 30 C. Accordingly, the 0.02 unit of the standardized solution employed in this assay should produce a change in optical density of 0.414 per minutes at 30 C. If such a change is produced by the foregoing assay, the reagent enzymes have sufficient activity to continue with the preparation of the described reagent.

To prepare the lyophilized powder for encapsulation, the following chemicals may be combined in the indicated ranges:

G. Tris-succinate buffer (prepared above) 100-250 Disodium creatine phosphate tetrahydrate 100-200 Magnesium sulfate, hydrated 80-120 Sodium adenosine-S-diphosphate dihydrate -30 TPN 12-18 Adenosine-S-minophosphate, monohydrate 60-120 Glucose, anhydrous 25-100 Cysteine hydrochloride 5-20 Lyophilized enzyme mixture 80-200 All of these chemicals are in the form of a dry powder and may be directly mixed together. The mixture is then reduced to a fine powder by any suitable means such as placing in a ball mill for an extended period of time. At this point the powder is ready for dividing into suitable units and packaged into capsules. In order to determine the size of each unit, the activity of the powder must first be determined. This is accomplished by dissolving a small sample of the specimen in a suitable quantity of water such as 2.9 ml. The assay test described above may then be repeated. By measuring the change of optical density produced by the assay reaction, the amount of activity of the enzymes in the powder may be determined. When this powder is dissolved in water it will make a reagent that will cause a reaction involving the adenosine triphosphate or ATP generated by the CPK reaction. This reaction will continue to increase in rate with increasing amounts of CPK until it is limited. The amount of CPK required for this to occur will be determined by the amount of activity of the reagent enzymes present. Accordingly, in order to compute the amount of the assay powder to be placed in each capsule, a convenient maximum amount of CPK for making the test must first be chosen. For example, this may be 0.05 unit. The size of the units into which the assay powder is divided will then be such that the rate of reaction will increase proportionally with the amount of CPK up to 0.05 unit or the change in optical density at 340 m for 0.05 unit should be 0.518 for 5 minutes at 30 C.

If it is desired to have all of the capsules of some predetermined size, a standard capsule size is selected that will always accommodate the lagrest unit of powder. After the size of the units is determined a suitable quantity of a bulking and stabilizing agent such as mannitol or its equivalent may be added to bring the volume to just the right size to fill the standard-sized capsule.

It may thus be seen that a large number of identical capsules will be produced that are suitable for assaying the amount of creatine phosphokinase or CPK present in a serum. The stabilizers tris-sulfate, acacia and ammonium sulfate will be effective to stabilize the enzymes and preserve their activity for extended periods of time. The tris-succinate buffer and the glucose substrate and TPN coenzyme will insure the assay reaction occurring.

To use one of the capsules to assay a serum to deter- 1 An International Enzyme Unit: is that amount ol. activity which will convert, one micromole of substrate per uunute at 30 C.

12 mine the amount of activity of adenosine triphosphate present in a serum, a suitable sample of the serum is first obtained. The optical density of the sample is first measured at a wavelength of 340 millimicrons. One of the capsules is then dissolved in a standard quantity of water such as 3 milliliters. This will create a liquid reagent of the correct size and activity for making one assay of serum sample. This liquid reagent may then be combined with the sample of serum whereby the following reactions will occur:

creatine phosphate+ADP Creatine phosphokinase creatiue-i-ATP;

glucose+ATP hexokinase glucose-6-P+ADP; g1ucose-6'P+TPN glucose-6-phosphate dehydrogenase fi-phoshogluconate-l-TPNH As these reactions progress, the ATP will form and then disappear and the TPN will be converted to TPNH. Since the components supplied by the reagent are in abundant supply, the only thing that will control the rate of the reaction will 'be the CPK present. It may thus be seen that the rate at which TPN is converted into TPNH will be a function of the quantity of the CPK present in the biological sample.

The reacions are allowed to continue for the duration of the normal test interval (5 to 10 minutes). However, before the expiration of this interval, the rate of reaction may be determined. At the end of the test interval, the optical density of the same is measured again at 340 millimicrons. Since the TPNH will absorb light at this wavelength, the difference between the optical densities before and after the reactions will be a result of the changes in the amount of the TPNH produced. Since the change in the quantity of TPNH with time is a function of the quantity of CPK originally present in the specimen this activity of CPK can be computed from the change in the optical density.

Adenosine monophosphate or AMP is added to the reagent mixture in order to reduce the activity of myokinase or adenylate kinase, which is at times present in biological samples. Myokinase would cause the following reaction to occur:

2 ADP myokinase ATP+AMP The ATP formed by this reaction would lead to false CPK determination; therefore, AMP is added which inhibits the formation of ATP by this reaction.

Cysteine, a disulfide compound, is added to protect or stabilize under some conditions the CPK activity. Other disulfide compounds may be employed with similar advantage, such as dithiothrietol (DTT), and dithioerythritrol (DTE).

EXAMPLE B In an alternaitve embodiment of the solid reagent for CPK determination, another thiol compound dithiothreitrol (Clelands Reagent) is employed with equally eflicacious results. Also a polyhydric substance is advantageously added during preparation for the afore discussed advantages of improving storage stability.

The resulting reagent fully prepared will consist of a dry mixture of the following solid substances.

enzymes: hexokinases and glucose-6-phosphate dehydrogenase buffer: 2-amino-2-methyl-1,3-propanediol and succinic acid stabilizer: acacia, tris (hydroxymethyl) sulfate aminomethane, ammonium sulfate and dithiothreitol substrates: creatine phosphate and glucose activator: magnesium aspartate coenzymes: adenosine diphosphate and triphosphospyridine nucleotide (TPN) inhibitor: adenosine monophosphate.

In order to prepare a large number of capsules of this reagent, the procedure described in the preceding example may be employed to produce a batch of dry reagent that may then be divided into small quantites and packaged into suitable capsules. The appropriate substitutions outlined a-bove result in a second solid reagent suitable for CPK determination.

For example, tablets prepared for a dilution up to 1.5 ml. of aqueous reagent typically have the following amounts.

Tris-(hydroxymethyl)-aminomethane-succinate 12.6 Mannitol 47.9 Optional lubricant 7.5

Total weight 97.55

What is claimed is:

1. A solid assay material for assaying a specimen for creatine kinase, comprising a solid, water soluble, substantially anhydrous, storage stable mixture of:

(a) the substrates creatine phosphate and glucose;

(b) the enzymes hexokinase and glucose-G-phosphate deydrogenase;

(c) the coenzymes comprising triphosphopyridine nucleotide and adenosine diphosphate;

(d) a buffer capable of maintaining the pH between 6.7 and 7.2;

(e) a source of magnesium ions;

(f) a stabilizer selected from the group consisting of mucilagenous gum, hydroxyalkylamines, ethylenediamine tetraacetic acid and its salt, a source of sulfate anion, and mixtures thereof; and

(g) the stabilizingbulking agent mannitol;

further wherein (b) and (f) are lyophilized mixture.

2. The material of claim 1 in which ((1) is tris (hydroxymethyl) aminomethane and succinic acid.

3. The material of claim 1 in which (d) is 2-amino- 2-methyl-1,3-propanediol and succinic acid.

4. The material of claim 1 in which (f) is acacia, tris (hydroxymethyl) aminomethane sulfate and ammonium sulfate.

5. The material of claim 1 further comprising a disulfide compound.

6. The material of claim 5 wherein the disulfide compound is cysteine.

7. The method of assaying a specimen for the enzyme creatine kinase using an assay material comprising a solid, water soluble, substantially anhydrous, storage stable mixture of:

(a) the substrate creatine phosphate and glucose;

(b) the enzymes hexokinase and glucose-6-phosphate dehydrogenase;

(c) the coenzymes triphosphopyridine nucleotide and adenosine diphosphate,

(d) a buffer capable of maintaining the pH between 6.7 and 7.2;

(e) a source of magnesium ions;

(f) a stabilizer selected from the group consisting of mucilagenous gums, hydroxylamines, ethylenediamine tetraacetic acid and its salts, a source of sulfate anion, and mixtures thereof; and

(g) the stabilizing-bulking agent mannitol;

further wherein (b) and (f) are a lyophilized mixture; which includes the steps of (i) dissolving in water said material thereby to produce a liquid reagent having a measurable optical density;

(ii) mixing said liquid reagent with said specimen to form a specimen-reagent assay mixture; and

(iii) measuring the rate of change of the optical density of the reacting specimen-reagent assay mixture.

8. The method of claim 7 in which (d) is tris (hydroxymethyl) aminomethane and succinic acid.

9. The method of claim 7 in which ((1) is 2-amino-2- methyl-1,3-propanediol and succinic acid.

10. The method of claim 7 in which (f) is acacia, tris (hydroxymetyhl) aminomethane sulfate, and ammonium sulfate.

11. The method of claim 7 further incorporating a disulfide compound in said assay material.

12. The method of claim 10 wherein the disulfide com- .pound is cysteine.

13. The method of claim 10 wherein the disulfide compound is dithiothreitol.

14. The material of claim 5 wherein the disulfide compound is dithiothreitol.

References Cited UNITED STATES PATENTS 1/1963 Innerfield l--63 5/1964 Puig l9563 Tanzer et al.: The Journal of Biological Chemistry, vol. 234, No. 12, pp. 3201-3204, December 1959.

ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 

